Malaria infects 200-400 million people each year causing 1-2 million deaths, thus remaining one of the most important infectious diseases in the world. Approximately 25 percent of all deaths of children in rural Africa between the ages of one and four years are caused by malaria. Due to the importance of the disease as a worldwide health problem, considerable effort is being expended to identify and develop malaria vaccines.
Malaria in humans is caused by four species of the parasite Plasmodium: P. falciparum, P. vivax, P. knowlesi and P. malariae. The major cause of malaria in humans if P. falciparum which infects 200 million to 400 million people every year, killing 1 to 4 million.
P. vivax (one of the four species infective to humans) cannot be cultured in vitro, as has been possible with P. knowlesi (a malarial strain found in old world monkeys which also invade human erythrocytes) and P. falciparum. Although P. vivax bears substantial phylogenetic similarity to P. knowlesi, the two species are different in many important respects. For example, P. vivax is not infective of many simian species and infection is poorly established in others, whereas P. knowlesi is poorly infective of humans while readily infecting many simian species.
The basis of various potential vaccines to combat malaria is appreciated through an understanding of the life cycle of the parasite. Infection in humans begins when young malarial parasites or xe2x80x9csporozoitesxe2x80x9d are injected into the bloodstream of a human by the mosquito. Following injection, the parasite localizes to liver cells. After approximately one week the parasites or xe2x80x9cmerozoitesxe2x80x9d are released into the bloodstream. The entry of the parasites into the bloodstream begins the xe2x80x9cerythrocyticxe2x80x9d phase. Each parasite enters the red blood cell in order to grow and develop. When the merozoite matures in the red blood cell, it is known as a trophozoite. The trophozoite undergoes several rounds of nuclear division (schizogony) until it ruptures the erythrocyte, releasing from 6 to 24 merozoites. After several asexual schizogonic cycles, some parasites, instead of becoming schizonts through asexual reproduction, develop into morphologically distinct forms known as xe2x80x9cgametocytesxe2x80x9d which are long-lived and undergo sexual development.
Sexual development of the malaria parasites involve the female or xe2x80x9cmacrogametocytexe2x80x9d and the male parasite or xe2x80x9cmicrogametocyte.xe2x80x9d These gametocytes do not undergo any further development in humans. Upon ingestion of the gametocytes into the mosquito, the complicated sexual cycle begins in the midgut of the mosquito. The red blood cells disintegrate in the midgut of the mosquito after 10 to 20 minutes. The microgametocyte continues to develop through exflagellation and releases 8 highly flagellated microgametes. Fertilization occurs upon fusion of the microgamete and the macrogamete. The fertilized parasite is known as a zygote which develops into an xe2x80x9cookinete.xe2x80x9d The ookinete embeds in the midgut of the mosquito, transforming into an oocyst within which many small sporozoites form. Before embedding in the midgut, the ookinete must first penetrate the peritrophic membrane which apparently acts as a barrier for invasion of ingested parasites. When the oocyst ruptures the sporozoites migrate to the salivary gland of the mosquito via the hemolymph. Once in the saliva of the mosquito, the parasite can be injected into a host.
The erythrocytic stage of the Plasmodium life cycle is of special relevance to vaccine development because the clinical and pathologic features of malaria in the host are attributable to this stage. In P. vivax, and P. knowlesi, Duffy blood group determinants present on Duffy positive erythrocytes are essential for invasion of human erythrocytes (Miller et al., Science 189: 561-563, (1975); Miller et al., N. Engl. J. Med. 295: 302-304, (1976)). In P. falciparum, invasion of merozoites into erythrocytes appears to be dependent on binding to sialic acids on glycophorins on the erythrocyte (Miller, et al., J. Exp. Med. 146: 277-281, (1971); Pasvol, et al., Lancet. ii: 947-950 (1982); Pasvol, et al., Nature, 279: 64-66 (1982); Perkins, J. Exp. Med. 160: 788-798 (1984)). Studies with the monkey parasite P. knowlesi allow a clearer understanding of the multiple events that occur during invasion. It is likely that even though P. vivax and P. falciparum bind to the Duffy antigen and sialic acids respectively, they share common strategies of invasion with each other and with P. knowlesi. 
In P. knowlesi, during invasion a merozoite first attaches to an erythrocyte on any surface of the merozoite, then reorients so that its apical end is in contact with the erythrocyte (Dvorak et al., Science 187: 748-750, (1975)). Both attachment and reorientation of merozoites occur equally well on Duffy positive and Duffy negative cells. A junction then forms between the apical end of the merozoite and the Duffy positive erythrocyte followed by vacuole formation and entry of the merozoite into the vacuole. Aikawa et al., J. Cell Biol. 77: 72-82 (1978). Junction formation and merozoite entry into the erythrocyte do not occur on Duffy negative cells (Miller et al., J. Exp. Med. 149: 172-184 (1979)), suggesting that a receptor specific for the Duffy determinant is involved in apical junction formation but not initial attachment.
The apical end of the merozoite is defined by the presence of three organelles: rhopteries, dense granules and micronemes. The rhopteries and dense granules release their contents at vacuole formation (Ladda et al., 1969; Aikawa et al., J. Cell Biol., 77: 72-82 (1978); Torn et al., Infection and Immunity 57: 3230-3233 (1989); Bannister and Dluzewski, Blood Cells 16: 257-292 (1990)). To date the function of the microneme is unknown. Nevertheless, the location of the micronemes suggest that they are involved in the invasion process. Duffy Antigen Binding Protein (DABP) and Sialic Acid Binding Protein (SABP) have been localized to the micronemes of P. knowlesi and P. falciparum respectively (Adams et al., Cell 63.: 141-153 (1990); Sim et al., Mol. Biochem. Parasitol. 51: 157-160. (1992)).
DABP and SABP are soluble proteins that appear in the culture supernatant after infected erythrocytes release merozoites. Immunochemical data indicate that DABP and SABP which are the respective ligands for the P. vivax and P. falciparum Duffy and sialic acid receptors on erythrocytes, possess specificities of binding which are identical either in soluble or membrane bound form.
DABP is a 135 kDa protein which binds specifically to Duffy blood group determinants (Wertheimer et al., Exp. Parasitol. 69: 340-350 (1989); Barnwell, et al., J. Exp. Med. 169: 1795-1802 (1989)). Thus, binding of DABP is specific to human Duffy positive erythrocytes. There are four major Duffy phenotypes for human erythrocytes: Fy(a), Fy(b), Fy(ab) and Fy(negative), as defined by the anti-Fya and anti-Fyb sera (Hadley et al., In Red Cell Antigens and Antibodies, G. Garratty, ed. (Arlington,. Va.:American Association of Blood Banks) pp. 17-33 (1986)). DABP binds equally to both Fy(a) and Fy(b) erythrocytes which are equally susceptible to invasion by P. vivax; but not to Fy(negative) erythrocytes.
In the case of SABP, a 175 kDa protein, binding is specific to the glycophorin sialic acid residues on erythrocytes (Camus and Hadley, Science 230:553-556 (1985); Orlandi, et al., J. Cell Biol. 116:901-909 (1992)). Thus, neuraminidase treatment (which cleaves off sialic acid residues) render erythrocytes, immune to P. falciparum invasion.
The specificities of binding and correlation to invasion by the parasite thus indicate that DABP and SABP are the proteins of P. vivax and P. falciparum which interact with sialic acids and the Duffy antigen on the erythrocyte. The genes encoding both proteins have been cloned and the DNA and predicted protein sequences have been determined (B. Kim Lee Sim, et al., J. Cell Biol. 111. 1877-1884 (1990); Fang, X., et al., Mol. Biochem Parasitol. 44: 125-132 (1991)).
Despite considerable research efforts worldwide, because of the complexity of the Plasmodium parasite and its interaction with its host, it has not been possible to discover a satisfactory solution for prevention or abatement of the blood stage of malaria. Because malaria is a such a large worldwide health problem, there is a need for methods that abate the impact of this disease. The present invention provides effective preventive and therapeutic measures against Plasmodium invasion.
The present invention provides compositions comprising an isolated DABP binding domain polypeptides and/or isolated SABP binding domain polypeptides. The DABP binding domain polypeptides preferably comprise between about 200 and about 300 amino acid residues while the SABP binding domain polypeptides preferably comprises between about 200 and about 600 amino acid residues. A preferred DABP binding domain polypeptide has residues 1 to about 325 of the amino acid sequence found in SEQ ID NO:2. A preferred SABP binding domain polypeptide has residues 1 to about 616 of the amino acid sequence of SEQ ID NO:4.
The present invention also includes pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an isolated DABP binding domain polypeptide in an amount sufficient to induce a protective immune response to Plasmodium vivax merozoites in an organism. In addition, isolated SABP binding domain polypeptide in an amount sufficient to induce a protective immune response to Plasmodium falciparum may be added to the pharmaceutical composition.
Also provided are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an isolated SABP binding domain polypeptide in an amount sufficient to induce a protective immune response to Plasmodium falciparum merozoites in an organism. In addition, isolated DABP binding domain polypeptide in an amount sufficient to induce a protective immune response to Plasmodium vivax may be added to the pharmaceutical composition.
Isolated polynucleotides which encode a DABP binding domain polypeptides or SABP binding domain polypeptides are also disclosed. In addition the present invention includes a recombinant cell comprising the polynucleotide encoding the DABP binding domain polypeptide.
The current invention further includes methods of inducing a protective immune response to Plasmodium merozoites in a patient. The methods comprise administering to the, patient an immunologically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an isolated DABP binding domain polypeptide, an SABP binding domain polypeptide or a combination thereof.
The present disclosure also provides DNA sequences from additional P. falciparum genes in the erythrocyte binding ligand (EBL) family that have regions conserved with the P. falciparum 175 kD and P. vivax 135 kD binding proteins.
As used herein a xe2x80x9cDABP binding domain polypeptidexe2x80x9d or a xe2x80x9cSABP binding domain polypeptidexe2x80x9d are polypeptides substantially identical (as defined below) to a sequence from the cysteine-rich, amino-terminal region of the Duffy antigen binding protein (DABP) or sialic acid binding protein (SABP), respectively. Such polypeptides are capable of binding either the Duffy antigen or sialic acid residues on glycophorin. In particular, DABP, binding domain polypeptides consist of amino acid residues substantially similar to a sequence of SABP within a binding domain from the N-terminal amino acid (residue 1) to about residue 325. SABP binding domain polypeptides consist of residues substantially similar to a sequence of DABP within a binding domain from the N-terminal amino acid (residue 1) to about residue 616.
The binding domain polypeptides encoded by the genes of the EBL family consist of those residues substantially identical to the sequence of the binding domains of DABP and SABP as defined above. The EBL family comprises sequences with substantial similarity to the conserved regions of the DABP and SABP. These include those sequences reported here as EBL-e1 (SEQ ID NO:5 and SEQ ID NO:6), E31a (SEQ ID NO:7 and SEQ ID No:8), EBL-e2 (SEQ ID NO:9 and SEQ ID NO:10) and Proj3 (SEQ ID NO:11 and SEQ ID NO:12).
The polypeptides of the invention can consist of the full length binding domain or a fragment thereof. Typically DABP binding domain polypeptides will consist of from about 50 to about 325 residues, preferably between about 75 and 300, more preferably between about 100 and about 250 residues. SABP binding domain polypeptides will consist of from about 50 to about 616 residues, preferably between about 75 and 300, more preferably between about 100 and about 250 residues.
Particularly preferred polypeptides of the invention are those within the binding domain that are conserved between SABP and the EBL family. Residues within these conserved domains are shown in FIG. 1, below.
Two polynucleotides or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues in the two sequences is the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482.(1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science: Dr., Madison, Wis.), or by inspection. These references are incorporated herein by reference.
The term xe2x80x9csubstantial identityxe2x80x9d means that a polypeptide comprises a sequence that has at least 80% sequence identity, preferably 90%, more preferably 95% or more, compared to a reference sequence over a comparison window of about 20 residues to about 600 residuesxe2x80x94typically about 50 to about 500 residues usually about 250 to 300 residues. The values of percent identity are determined using the programs above. Particularly preferred peptides of the present invention comprise a sequence in which at least 70% of the cysteine residues conserved in DABP and SABP are present. Additionally, the peptide will comprise a sequence in which at least 50% of the Tryptophan residues conserved in DABP and SABP are present. The term substantial similarity is also specifically defined here with respect to those amino acid residues found to be conserved between DABP, SABP and the sequences of the EBL family. These conserved amino acids consist prominently of tryptophan and cysteine residues conserved among all sequences reported here. In addition the conserved amino acid residues include phenylalanine residues which may be substituted with tyrosine. These amino acid residues may be determined to be conserved after the sequences have been aligned using methods outlined above by someone skilled in the art.
Another indication that polypeptide sequences are substantially identical is if one protein is immunologically reactive with antibodies raised against the other protein. Thus, the polypeptides of the invention include polypeptides immunologically reactive with antibodies raised against the SABP binding domain, the DABP binding domain or raised against the conserved regions of the EBL family.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60xc2x0 C.
The phrases xe2x80x9cisolatedxe2x80x9d or xe2x80x9cbiologically purexe2x80x9d refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. Thus, the binding domain polypeptides of this invention do not contain materials normally associated with their in situ environment, e.g., other proteins from a merozoite membrane. However, even where a protein has been isolated to a homogenous or dominant band by PAGE, there can be trace contaminants in the range of 5-10% of native protein which co-purify with the desired protein. Isolated polypeptides of this invention do not contain such endogenous co-purified protein.
Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.
The term xe2x80x9cresiduexe2x80x9d refers to an amino acid (D or L) or amino acid mimetic incorporated in a oligopeptide by an amide bond or amide bond mimetic. An amide bond mimetic of the invention includes peptide backbone modifications well known to those skilled in the art.